Repression of mRNA translation initiation by GIGYF1 via disrupting the eIF3-eIF4G1 interaction

Viruses can selectively repress the translation of mRNAs involved in the antiviral response. RNA viruses exploit the Grb10-interacting GYF (glycine-tyrosine-phenylalanine) proteins 2 (GIGYF2) and eukaryotic translation initiation factor 4E (eIF4E) homologous protein 4EHP to selectively repress the translation of transcripts such as Ifnb1, which encodes the antiviral cytokine interferon-β (IFN-β). Herein, we reveal that GIGYF1, a paralog of GIGYF2, robustly represses cellular mRNA translation through a distinct 4EHP-independent mechanism. Upon recruitment to a target mRNA, GIGYF1 binds to subunits of eukaryotic translation initiation factor 3 (eIF3) at the eIF3-eIF4G1 interaction interface. This interaction disrupts the eIF3 binding to eIF4G1, resulting in transcript-specific translational repression. Depletion of GIGYF1 induces a robust immune response by derepressing IFN-β production. Our study highlights a unique mechanism of translational regulation by GIGYF1 that involves sequestering eIF3 and abrogating its binding to eIF4G1. This mechanism has profound implications for the host response to viral infections.

cells were transfected with psiCHECK2-RL-Ifnb1 3´ UTR reporter or the psiCHECK2 reporter (as control).RL and FL activities were measured 24 h after transfection.The RL/FL ratio in psiCHECK2-RL-Ifnb1 3´ UTR reporter-expressing cells was normalized to the psiCHECK2expressing cells.Data are presented as mean ± SD (n=3).ns = non-significant, ****P < 0.0001; two-way ANOVA with Bonferroni's post-hoc test.(B) Western blot analysis of cell lysate from Figure 2C with the indicated antibodies.(C & D) Tether-function assays for measurement of repression of the deadenylation-permissive RL-5boxB-polyA (C) or deadenylation-resistant RL-5BoxB-HhR reporters (D) upon coexpression with increasing amounts of λN-v5-GIGYF1 or λN-v5-GIGYF2 plasmids in HEK293 cells.Data are presented as mean ± SD (n=3).*P < 0.05, ****P < 0.0001; two-way ANOVA with Bonferroni's post-hoc test.(E-F) Western blotting with the indicated antibodies using lysates from cells shown in Figure 2E and 2F, respectively.(G) Tetherfunction assay with the full-length, N-terminal (aa1-417), and C-terminal (aa541-1035) fragments of GIGYF1 in GIGYF1-KO HEK293 cells.Cells were co-transfected with the indicated plasmid along with the deadenylation-resistant RL-5BoxB-HhR and FL control plasmid, followed by dualluciferase measurement assay 24 h post-transfection.Data are presented as mean ± SD (n=3).ns = non-significant, ****P < 0.0001; one-way ANOVA with Bonferroni's post-hoc test.(H) Western blotting with the indicated antibodies using lysates from cells shown in (G).(I) ELISA measurement of IFN-β production in GIGYF1-KO HEK293 cells overexpressing the full-length GIGYF1 and the indicated truncated isoforms following 6 h of treatment with 1 μg/mL poly(I:C).Data are presented as mean ± SD (n=3).ns = non-significant, ****P < 0.0001; one-way ANOVA with Bonferroni's post-hoc test.(J) Western blotting with the indicated antibodies using lysates from cells shown in (I).(K) Western blotting with the indicated antibodies using lysates from cells shown in Figure 2H.A-C) Co-IP assay for detecting the interaction of FLAG-eIF3E (A), FLAG-eIF3D (B), or FLAG-eIF3G (C) with v5-GIGYF1 or v5-GIGYF2 in HEK293T cells.Whole-cell lysates were prepared 24 h after transfection and subjected to immunoprecipitation using an anti-FLAG antibody followed by blotting with the indicated antibodies.(D) Left: PLA for detection of GIGYF1-eIF3D interaction in HEK293T cells transfected with vectors expressing v5-GIGYF1 or v5-GIGYF2 along with FLAG-eIF3D.24 h post-transfection cells were fixed and subjected to PLA using FLAG and v5 antibodies.Scale bar = 10 µm.Right: The bar graphs represent the number of PLA signals from at least 30 cells, counted in each sample.n=5 independent experiments.Data are presented as mean ± SD. ns= non-significant, ****P< 0.0001; unpaired t-test.(E) Left: PLA for detection of GIGYF1-eIF3G interaction in HEK293T cells transfected with vectors expressing v5-GIGYF1 or v5-GIGYF2 along with FLAG-eIF3G.24 h post-transfection cells were fixed and subjected to PLA using FLAG and v5 antibodies.Scale bar = 10 µm.Right: The bar graphs represent the number of PLA signals from at least 30 cells, counted in each sample.n=5 independent experiments.Data are presented as mean ± SD. ns= non-significant, ****P< 0.0001; unpaired t-test.(F-I) Western blot analysis of the samples in Figure 3B, Figure 3C, Supp. Figure 4D, and Supp. Figure 4E respectively with the indicated antibodies.(J) Fractionation of endogenous GIGYF1, GIGYF2, and the indicated subunits of eIF3 complex by size-exclusion chromatography.A total of 10 mg of proteins from HEK293 cells was loaded onto a Superose 6 column and ran at a flow rate of 0.4 mL/min.Fractions of 0.5 mL were collected, and 50 μL of each fraction was subjected to western blot analysis.The elution position of the molecular size markers is shown.(K) Schematic of the domain structures of full-length (FL) GIGYF1 and truncated isoforms A-E used in Figure 3D and Supp. Figure 5A.(L) Western blot analysis of the samples in Figure 3D with the indicated antibodies.(M) Western blot analysis of the samples in Figure 3E with the indicated antibodies.Whole-cell lysates were prepared 24 h after transfection and subjected to immunoprecipitation using eIF3D antibody followed by blotting with the indicated antibodies.(B) Co-IP assay for detection of interactions of FLAG-eIF3D with eIF2α and TTP in GIGYF1-KO cells expressing v5-GIGYF1 or v5-Empty control plasmids.Whole-cell lysates were prepared 24 h after transfection and subjected to immunoprecipitation using FLAG antibody followed by blotting with the indicated antibodies.(C) Co-IP assay for detection of interactions of eIF3D with eIF3E and eIF4G1 in GIGYF1-KO cells expressing increasing amounts of GIGYF1.Whole-cell lysates were prepared 24 h after transfection and subjected to immunoprecipitation using anti-eIF3D antibody followed by blotting with the indicated antibodies.(D) Co-IP assay for detection of interactions of   Data S2.Gene Ontology analysis of the BirA*-GIGYF1 proximal proteins identified with BioID assay in HEK293 cells.Gene Ontology analysis of the significant proximity interactors (FDR<0.01)was performed using the online g:profiler tool: https://biit.cs.ut.ee/gprofiler/gost.

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Fig. S5.The C-terminal region of GIGYF1 mediates its interactions with eIF3D; related to Figure 3. (A) Left: HEK293T cells were co-transfected with FLAG-eIF3D and the full-length or indicated GIGYF1 truncation mutants (see Supp.Fig.4K for more details) or full-length GIGYF1 (as control).24 h post-transfection cells were fixed and subjected to PLA using FLAG and v5 antibodies.PLA signals are shown in yellow.The nucleus and actin cytoskeleton were counterstained with DAPI and phalloidin (red), respectively.Scale bar = 10 µm.Right: The bar graphs represent the number of PLA signals from at least 20 cells, counted in each sample.n=3 independent experiments.ns = non-significant, ****P< 0.0001; one-way ANOVA with Bonferroni's post-hoc test.(B) Western blot analysis of cell lysates from (A) with the indicated antibodies.(C) Left: PLA assay for detection of the interactions between eIF3D and the indicated two chimeric constructs described in Figure 2G.Right: The bar graphs represent the number of PLA signals from at least 20 cells, counted in each sample.n=3 independent experiments.Data are presented as mean ± SD. ns= non-significant, ****P < 0.0001; one-way ANOVA with Bonferroni's post-hoc test.(D) Western blot analysis of cell lysates from (C) with the indicated antibodies.

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Fig. S6.Ectopic expression of GIGYF1 reduces interactions of endogenous eIF3 and eIF4G1 proteins; related to Figure 4. (A) Co-IP assay for assessing the impact of GIGYF1 on interaction of eIF3D with eIF3E, eIF4G1, and eIF4G2.GIGYF1-KO HEK293 cells were transfected with vectors expressing the v5-tagged full-length or N-terminal fragment (aa1-239) of GIGYF1.Whole-cell lysates were prepared 24 h after transfection and subjected to immunoprecipitation using eIF3D antibody followed by blotting with the indicated antibodies.(B) Co-IP assay for detection of interactions of FLAG-eIF3D with eIF2α and TTP in GIGYF1-KO cells expressing v5-GIGYF1 or v5-Empty control plasmids.Whole-cell lysates were prepared 24 h after transfection and subjected to immunoprecipitation using FLAG antibody followed by blotting with the indicated antibodies.(C) Co-IP assay for detection of interactions of eIF3D with eIF3E and eIF4G1 in GIGYF1-KO cells expressing increasing amounts of GIGYF1.Whole-cell lysates were prepared 24 h after transfection and subjected to immunoprecipitation using anti-eIF3D antibody followed by blotting with the indicated antibodies.(D) Co-IP assay for detection of interactions of

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Fig. S7.C-terminal truncated isoforms of GIGYF1 do not affect eIF4G1-eIF3D interaction; related to Figure 4. (A) Western blot analysis of cell lysates from (B). (B) PLA assay for detection of HA-eIF4G1 and FLAG-eIF3D in GIGYF1-KO cells transfected with v5-GIGYF1 or truncated isoforms of GIGYF1 (aa1-239, aa541-839 and aa829-1035).(C) Western blot analysis of cell lysates from (D). (D) PLA assay for detection of HA-eIF4G1 and FLAG-eIF3D in GIGYF1-KO cells transfected with aa1-239 or aa541-1035 truncated isoforms of GIGYF1.24 h posttransfection cells were fixed and subjected to PLA using HA and FLAG antibodies.The bar graphs represent the number of PLA signals from at least 30 cells, counted in each sample.n=3 independent experiments.****P< 0.0001; unpaired t-test.

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Fig. S8.GIGYF1 abrogates eIF3/eIF4G1 interaction upon poly(I:C) stimulation; related to Figure 5. (A) Co-IP assay to detect the interaction of endogenous eIF3L, eIF3E, and eIF4G1 upon poly(I:C) stimulation in GIGYF1-KO cells.Whole-cell lysates were prepared 24 h after transfection and subjected to immunoprecipitation using anti-eIF3L antibody followed by blotting with the indicated antibodies.(B) Left: PLA assay for detection of interactions between v5-GIGYF1 and FLAG-eIF3G upon poly(I:C) stimulation.PLA signals are shown in yellow, the nucleus and Actin cytoskeleton were counterstained with DAPI and phalloidin (red), respectively.Scale bar = 10 µm.Right: The bar graphs represent the number of PLA signals from at least 20 cells, counted in each sample.n=3 independent experiments.Data are presented as mean ± SD. ns= non-significant, ****P< 0.0001; unpaired t-test.(C) Western blot analysis of cell lysates from (B). (D) Western blot analysis of the indicated protein in the v5-GIGYF1 RIP assay (Figures 5I).v5-GIGYF1 was immunoprecipitated using a v5 antibody in vehicle and poly(I:C)-treated HEK293 cells.Precipitated proteins were separated by SDS-PAGE and probed with the specified antibodies.
Fig. S8.GIGYF1 abrogates eIF3/eIF4G1 interaction upon poly(I:C) stimulation; related to Figure 5. (A) Co-IP assay to detect the interaction of endogenous eIF3L, eIF3E, and eIF4G1 upon poly(I:C) stimulation in GIGYF1-KO cells.Whole-cell lysates were prepared 24 h after transfection and subjected to immunoprecipitation using anti-eIF3L antibody followed by blotting with the indicated antibodies.(B) Left: PLA assay for detection of interactions between v5-GIGYF1 and FLAG-eIF3G upon poly(I:C) stimulation.PLA signals are shown in yellow, the nucleus and Actin cytoskeleton were counterstained with DAPI and phalloidin (red), respectively.Scale bar = 10 µm.Right: The bar graphs represent the number of PLA signals from at least 20 cells, counted in each sample.n=3 independent experiments.Data are presented as mean ± SD. ns= non-significant, ****P< 0.0001; unpaired t-test.(C) Western blot analysis of cell lysates from (B). (D) Western blot analysis of the indicated protein in the v5-GIGYF1 RIP assay (Figures 5I).v5-GIGYF1 was immunoprecipitated using a v5 antibody in vehicle and poly(I:C)-treated HEK293 cells.Precipitated proteins were separated by SDS-PAGE and probed with the specified antibodies.